answer to I've been approached by an investigator who wants to separate mesophyl chloroplasts from bundle sheath chloroplasts in maize, and this is way out of my area of expertise. They gave me a reference with Bob Ashcroft's name on it, so I was hoping to get some advice from the expert! The papers were fairly old and detailed several methods including the use of differences in scatter and in fluorescence emission in the 685-710 range to isolate them. They also used ratios of fluorescence above and below 710, which seems to relate to the emission difference between chlorophylls A and B. Is this necessary (as we have no filter currently to do this) or is there another way to do this? Is anyone out there doing something similar? Carol Oxford Manager, UC Davis Optical Biology Lab Tupper Hall, rm 3439 University of California Davis, California 95616 (530) 752-7205 (530) 752-4548 fax (530) 523-0967 pager I have been publishing onthat subject a long time ago. I link somes references below. 1. Schröder W.P. and Petit P.X. (1992) Flow cytometry of spinach chloroplasts: determination of the intactness and lectin-binding properties of the enbvelope and thylakoid membranes. Plant Physiol. 100 : 1092-1102. 2. Kausch, A.P., T.P. Owen, Jr., S. Narayanswami, and B.D. Bruce. 1999. Organelle isolation by magnetic immunoabsorption. Biotechniques 26: 336-43. 3. Pfundel, E., and A. Meister. 1996. Flow cytometry of mesophyll and bundle sheath chloroplast thylakoids of maize (Zea mays L.). Cytometry 23: 97-105. 4. Wolters, A.A., A.C. Vergunst, F. van der Werff, and M. Koornneef. 1993. Analysis of nuclear and organellar DNA of somatic hybrid calli and plants between Lycopersicon spp. and Nicotiana spp. Mol Gen Genet 241: 707-18. In fact, the idea of separating mesophyll and bundle sheath chloroplasts and mitochondria is an old idea... Which was supposed to be an alternative to the clasical preparation and purification on percoll gradients. But is it realistic to do so?.... The purification of chloroplast of both origin in an very intact conditions appears to me very problematic and the flow cytometry approach will make you very desapointed because the technology is not fair in the sense that she will tell you the exact quality of your preparation. Anyway, if you find somes informations in the 1992 paper, just E-mail me and I will help you as much as possible Petit P.X. and Cantrel C. (1986) Mitochondria from Zea mays leaf tissues: differentiation of carbon assimilation and photorespiratory activity between mesophyll and bundle sheath cells. Physiol. Plant. 67 : 442-446. Petit P.X. (1984) Mitochondria from the mesophyll cells of Zea mays leaves. J. Plant Physiol. 116 : 351-364. Sincerely yours Patroce \ | / (o o) \ | / _______________oOo_____oOo_____oOo_ (o o)__oOo________________ Dr. Petit Patrice X. Institut Cochin de Génétique Moléculaire INSERM U129 - CHU Cochin Port-Royal 24, rue du Faubourg Saint-Jacques F-75014 Paris, France. Tel: 33 01 44 41 24 11 Fax: 33 01 44 41 24 21 E-mail: pxpetit@icgm.cochin.inserm.fr
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